A Protein from a Parasitic Microorganism Rickettsiae Can Cleave the

Signal Sequences of Proteins Targeting Mitochondria

Sakae Kitada1*, Tsuneo Uchiyama2, Tomoyuki Funatsu1, Yumiko Kitada1, Tadashi Ogishima1,

and Akio Ito1

From 1Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka

812-8581, Japan and 2Department of Virology, Institute of Health Biosciences, The

University of Tokushima Graduate School, Tokushima 770-8503, Japan.

Running title: Evolution of Mitochondrial Preprotein Processing *Corresponding auther. Mailing address: Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan. Tel.: +81-92-642-4182; Fax: +81-92-642-2607; E-mail: s.kitscc@mbox.nc.kyushu-u.ac.jp

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Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01261-06 JB Accepts, published online ahead of print on 8 December 2006

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ABSTRACT

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria

than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found

to resemble the c and d subunits of mitochondrial processing peptidase (MPP), which cleaves

the transport signal sequences of mitochondrial preproteins. RPP showed completely

conserved zinc-binding and catalytic residues compared with d-MPP, but barely contained any

of the glycine-rich loop region characteristic of c-MPP. When the biochemical activity of RPP

purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides

and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover,

RPP appeared to activate yeast! d-MPP so that it processed preproteins with shorter

presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide

peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These

are the first biological and enzymological studies to report that a protein from a parasitic

microorganism can cleave the signal sequences of proteins targeted to mitochondria.

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INTRODUCTION

The endosymbiont hypothesis for the origin of mitochondria in eukaryotes is now

widely accepted, along with a growing interest in evolution as entire genomic sequences

are revealed from various organisms. According to this hypothesis, a free-living

bacterium as an organelle progenitor once entered an anaerobic organism, which is thought

to be an archaebacterial ancestor, and established a constitutive endosymbiotic relationship

with the host cells, mainly to supply ATP (1-3). Modern mitochondria originated when

the parasitic invader lost most of its own genome and began to depend on nuclear-encoded

proteins for its biogenesis, although it retained control of the eukaryotic cell viability via

metabolic and apoptotic pathways.

Most mitochondrial proteins are encoded in the nucleus and synthesized by

cytoplasmic ribosomes as preproteins with N-terminal presequences that are required for

targeting to mitochondria (4-5). These preproteins are unfolded and imported into the

mitochondrial matrix across the double membrane through protein translocation machinery

comprising a translocase on the outer mitochondrial membrane (Tom) and a translocase on

the inner mitochondrial membrane (Tim) (6-9). Finally, the presequences are cleaved by a

matrix-located metalloendopeptidase, i.e., mitochondrial processing peptidase (MPP)

(10-12). Overall, this proteolytic processing is involved in maturation of the

mitochondrial proteins and is essential for eukaryotic cell viability from unicellular (13) to

multicellular (14) organisms. Therefore, the c and d subunits of MPP (c- and d-MPP,

respectively) tightly regulate the protease action and specifically cleave the preproteins.

The genome sequences of the obligate intracellular parasitic bacteria rickettsiae (the

agents that cause typhus) reveal strikingly similar gene profiles to those of mitochondria

(15-17). Among the bacteria examined to date, rickettsiae are more closely related to

mitochondria than any other bacteria analyzed at the genome level. Interestingly,

Rickettsia prowazekii gene 219 (RP219) encodes a putative peptidase (rickettsial putative

protease, RPP) that is highly similar to MPPs (15), and corresponding genes have also been

found in other rickettsial species (16-17). Similar to d-MPPs, which are the catalytic MPP

subunits (18-19), RPPs have a zinc-binding motif, HxxEHx75E, in which the histidine

residues and final glutamate residue presumably participate in metal-binding, while the first

glutamate residue could be involved in water-activation for hydrolysis of the peptide bond.

These active site motifs are found in the M16 protease family of metalloendopeptidases

characterized by Escherichia coli pitrilysin and insulinases from mammal and insects,

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which appear to diverge widely from bacteria to higher eukaryotes (20).

In the present study, we analyzed the structural characteristics and biochemical

activities of the RPP from R. prowazekii. The RPP primary structure resembled both the

MPP subunits, since the N- and C-terminal regions of RPP were similar to the N-domains

of d-MPPs and C-domains of c-MPPs, respectively. The biphasic structure of RPP

seemed to reflect dual functions, namely catalytic and regulatory actions toward basic

peptides and preprotein processing, respectively. Thus, the characteristics of the

structures and functions of RPP and the MPP subunits could be inherited from a common

ancestor protein in the parasite that led the endosymbiotic evolution of mitochondria.

Here, we discuss the evolutionary and functional relationships between proteins that

resemble components of the mitochondrial transport-processing system on the basis of

mitochondrial endosymbiotic evolution.

MATERIALS AND METHODS

Genetic analyses - The amino acid sequences of MPP and RPP were assembled from

the Swiss-Prot and Genome databases, and aligned using the CLUSTAL W program (21).

From this multiple sequence alignment, a consensus sequence for the active site of MPP

was derived. The phylogeny was inferred using the bootstrap method with 1000 trials and

a bootstrap tree was drawn using TreeView 1.6.6. Genomic blastp searches to identify

microbial MPP-like proteins were carried out using the BLAST website for microbial

genomes at the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi), using the whole protein sequence of

yeast c or d-MPP. The searches were performed with the EXPECT parameter set to

0.0001 and the FILTER set to default. In the resultant proteins, we collected MPP-like

proteins with expected values under 1xE-10 and confirmed whether each sequence had a

GRL region.

Culture of rickettsiae and RPP expression analyses – R. typhi, R. conorii and

Rickettsia japonica were grown in Vero cells at 34ºC and purified as described previously

(22). Total RNA was extracted from purified R. typhi cells using a total RNA isolation

system (Promega) according to the manufacturer’s instructions. Next, cDNAs were

synthesized from the total RNA using reverse transcriptase (Promega) and the primers

5’-TTGGATCCTTAAAATCCATTAAGATCATTC-3’ for rpp and

5’-TTAGAAGTCCATACCACC-3’ for groEL. Amplification of the DNA fragments was

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performed by PCR using the primer pairs 5’-ATGATAATCCTGATGACC-3’ and

5’-TTCTTCTGACTTATAGG-3’ for rpp and 5’-CTTCAAGAGCGTTTAGC-3’ and

5’-TATCAGAAGGTTCATCC-3’ for groEL. Proteins from purified rickettsiae were

separated by SDS-PAGE and analyzed by western blotting using a rabbit anti-RPP antibody

and horseradish peroxidase-conjugated goat anti-rabbit IgG.

Overproduction, purification and characterization of recombinant proteins - An R.

prowazekii genomic clone including RP219 inserted into lambda-Zap (a kind gift from Drs.

Anderson and Kurland (University of Uppsala)) was used as the source of the RPP gene.

The DNA fragment for the open reading frame of RPP was amplified by PCR using an

RPP-specific pair of 5’- and 3’-primers and ligated into the expression vector pET-23d

(Novagen), which produces proteins with a C-terminal His6-tag, thereby constructing

pET-RPP. E. coli BL21 cells were cotransformed with pET-RPP and pKY206, a plasmid

encoding the E. coli GroE operon. After culturing the cells in LB medium at 30ºC for 24

h, the proteins were extracted and purified using a nickel-chelating Sepharose (Amersham

Biosciences) column as described previously (19). The RPP-containing fractions eluted

from the affinity column were pooled and incubated with 5 mM ATP at 0ºC for 60 min to

release the GroEL and ES associated with RPP. The protein solution was diluted by more

than 10-fold with buffer A (20 mM Tris-HCl pH 7.5, 30% glycerol and 0.01% Tween-20)

and loaded onto a DEAE Sepharose (Amersham Biosciences) column equilibrated with

buffer A. After washing the column, RPP was eluted with buffer A containing 100 mM

NaCl. The last purification step was repeated once. Size exclusion chromatography was

performed, using TSK-GEL SUPER SW 3000 (TOSOH) in 20 mM Tris-HCl (pH 7.5)

containing 100 mM NaCl. The gene for PRR* (E52Q mutant) was engineered using a

QuikChange site-directed mutagenesis system (Promega) and the protein was produced and

purified as described above. Wild-type MPP, d-MPP* (E73Q mutant) and c-MPPFGRL

(F287-249 mutant) were purified as described previously (19, 23).

Assay for peptidase activity and identification of cleavage sites - The peptides

purchased for this study were dynorphin A (Peninsula Laboratories Inc.), vasoactive

intestinal peptide (Peninsula Laboratories Inc.), mastoparan (Peninsula Laboratories Inc.),

c melanocyte stimulating hormone (cMSH) amide (C S Bio Co.), big endothelin 1

(Phoenix Pharmaceuticals Inc.), peptide fragment of somatostatin receptor SSTR2A

(Gramsch Laboratories), peptide fragment of o opioid receptor MOR1A (Gramsch

Laboratories) and gastrin I (Bachem). We also used various presequence peptides

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synthesized as described previously (24): mouse malate dehydrogenase (MDH2-28), yeast

heat shock protein SSC1 (HS771-32), yeast MDH2-17, mouse aldehyde dehydrogenase

(ALDH) 1-29, human ornitine aminotranserase (OAT1-25), yeast ubiquinol cytochrome c

reductase subunit2 (UCR21-32), bovine adrenodoxin (ADX18-57) and yeast cytochrome c

oxidase subunit4 (COXIV2-25). Each peptide (1 oM) was incubated with RPP (25 nM) or

yeast MPP (2.5 nM) at 30ºC for 60 min, before the reaction was stopped and analyzed by

reverse-phase HPLC as described previously (24). The cleavage efficiencies were

determined from the ratios of the residual peptide amounts after incubation with or without

peptidases. To define the cleavage sites, the eluates containing peptide fragments were

independently collected, lyophilized, mixed with a matrix solution of sinapinic acid and

analyzed using a mass spectrometer (VoyagerTM PR-HR Biospectrometry; ABI).

Assay for preprotein processing - Processing assays of [35S]methionine-labeled

preproteins were performed as described previously (19). RPP, MPP or premixed

combinations of RPP and MPPs were incubated with the preprotein at 30ºC for 60 min in

the processing buffer. The processing products were separated by SDS-PAGE and

visualized using an Imaging Analyzer (Bas1000; Fuji Film). The processing efficiency

was determined by quantifying the radioactivity of the cleaved protein relative to the total

protein using Image Gauge 3.0 (Fuji Photo Film Co. Ltd. and Koshin Graphic Systems).

RESULTS

Evolutionary and Structural Characteristics of RPP - RPPs were identified in the

genomes of several rickettsial species and found to be highly conserved with each other.

When three RPPs from R. prowazekii, R. typhi and R. conorii were compared with MPP

subunits from protozoa to mammals using statistical genetics, the potential phylogenetic

relationship between them revealed that RPPs were genetically close to either c-MPP and

d-MPP and more closely related to protozoan MPPs than other eukaryotic MPPs (Fig. 1).

Figure 2A shows structural schematic diagrams of R. prowazekii RPP and the yeast

Saccharomyces cerevisiae MPP subunits. It was of interest to note that the N- and

C-terminal regions of the RPP resembled the terminal domains of d-MPP and c-MPP (N

and C domains), respectively. In particular, the N-terminal region of the RPP included an

active site motif, HxxEHx75E, that was previously characterized in a subfamily of M16

proteases. Moreover, the rickettsial amino acid sequences around the motif were highly

conserved in the active site sequences of d-MPPs (Fig. 3), indicating that RPPs potentially

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possess peptidase activity. On the other hand, the glycine-rich loops (GRLs) that are

typically preserved in the C domains of c-MPPs were mostly lacking in the C domains of

RPPs, whereas the rickettsial amino acid sequences corresponding to regions around the

GRLs were comparatively similar to the primary structures of c-MPPs (Fig. 3), suggesting

a lesser importance for the GRL function among RPPs. The three-dimensional structure

of yeast MPP complexed with a synthetic presequence peptide is represented in Fig. 2B

according to the coordination data reported by Taylor et al. (25). Each yeast MPP subunit

contains N and C domains of ~210 residues with nearly identical folding topologies, that

are related by an approximate two-fold rotation. Thus, the higher-order structure of RPPs

appears to resemble those of the MPP subunits, and is probably more similar to d-MPP,

such that RPPs would be expected to show peptidase activity.

Gene and protein expression of RPP – We were Since R. prowazekii is unavailable in

Japan, we used other rickettsial species, R. typhi, of which RPP gene is closely related to R.

prowazekii one (Fig. 1). The RPP gene expression in R. typhi infecting simian cells was

examined by RT-PCR. Reverse-transcripts from R. typhi total RNA extracts were

amplified using specific primer pairs designed for the RPP-coding region, while RT-PCR

for the GroEL-coding region served as a control. DNA fragments of 680 and 500 bp for

the RPP and GroEL genes, respectively, were amplified from the transcripts in a reverse

transcriptase-dependent manner (Supplemental Fig. 1A). The DNA sequences of these

fragments revealed that the RT-PCR products were the same as the corresponding part of

each gene, indicating the existence of RPP mRNA in parasitic rickettsial cells. Three

species of rickettsiae appeared to produce RPP proteins (Supplemental Fig. 1B).

Immunoreactive proteins of around 50 kDa were detected although the rickettsial proteins

were slightly smaller than the recombinant RPP, probably due to the additional C-terminal

hexahistidine (His6) tag. To characterize the RPP, we tried to purify the recombinant

protein from an extract of E. coli cells transformed with a protein expression vector

carrying the RPP gene. After centrifugation of the cell lysate, the RPP was barely

recovered in the soluble protein fraction (Supplemental Fig. 1C). However, we succeeded

in producing the soluble protein by coexpression of the E. coli molecular chaperonins

GroEL and GroES (Supplemental Fig. 1C). The recombinant RPP was purified using a

combination of nickel affinity and ion exchange chromatographies and the protein

preparation was estimated to show >99% purity by SDS-PAGE and protein staining

(Supplemental Fig. 1C). Size exclusion chromatography revealed that the non-denatured

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molecular size was approximately 50 kDa (data not shown), suggesting that the

recombinant RPP was in a monomeric state.

Peptidase activity of RPP - Since RPPs showed conservation of the active site

sequence of metalloendopeptidases of the M16 protease family, we expected that the

recombinant RPP would show proteolytic activity. To investigate whether RPP inherently

hydrolyzes bound peptides, several synthetic peptides were incubated with the purified RPP

in vitro and analyzed by reverse-phase HPLC. Basic peptides from various sources,

namely dynorphin A, vasoactive intestinal peptide, mastoparan and cMSH amide,

produced detectable amounts of fragments (Table 1 and Supplemental Fig. 2) following

incubation with RPP, whereas cleavage of neutral and acidic peptides (peptides E to H in

Table 1) by RPP was undetectable. Thus, RPP showed preferential cleavage of the more

basic peptides among the peptides investigated. Mass spectrometry was carried out to

determine the molecular masses of the peptide fragments and reveal the cleavage sites.

RPP frequently hydrolyzed peptide bonds before hydrophobic residues and sometimes

attacked sites beside basic residues (Table 1). These experiments demonstrated that RPP

has intrinsic peptidase activity for basic peptides. Next, we further investigated the

proteolytic activity of RPP. Due to its preferential cleavage of basic sequences, the typical

basic proteins lysozyme and cytochrome c were tested under native and denaturing

conditions. RPP showed no proteolytic activity toward the proteins either in

non-denaturing buffer or under intensive denaturing conditions with urea when the proteins

were analyzed by SDS-PAGE and stained (Supplemental Fig. 3). After the RPP reactions,

the proteins were analyzed by reverse-phase HPLC, and the elution time of the lysozyme

solution that had been under the denaturing conditions was delayed compared to that for

the non-denatured lysozyme (data not shown), suggesting that the substrate protein was

unfolded during the RPP action. The remaining urea in the RPP reaction solution did not

affect the peptidase activity (data not shown). These results imply that RPP alone behaves

as a basic peptide peptidase in organisms.

Cleavage of mitochondrial presequences by RPP - RPP resembles the catalytic

subunit of MPPs, attacks basic sequences and particularly cleaves bonds before

hydrophobic residues and around basic residues, which MPPs also prefer. These insights

into the relationship between RPP and MPPs regarding their structures and substrate

specificities led us to investigate whether RPP cleaves mitochondrial presequences. When

a 16-residue presequence peptide, which included the normal presequence cleavage site

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between the residues Ala-8 and Phe-9, derived from the preprotein of yeast malate

dehydrogenase (MDH2-17) were incubated with RPP and analyzed by HPLC and mass

spectrometry, it was found that RPP cleaved one site between the alanine and

phenylalanine residues that was also processed by yeast MPP (Table 2 and Supplemental

Fig. 4). Since an RPP mutant in which the glutamate residue in the HxxEH motif was

substituted with glutamine (RPP*) was inactive (Supplemental Fig. 5), the peptidase

activity was specific and depended on the glutamate residue. RPP also cleaved other

synthetic presequence peptides of various lengths from different organisms at the one or

more sites, with the exception of the yeast COX IV2-25 peptide (Table 2). Among the 8

peptides tested, RPP attacked 5 of them at peptide bonds corresponding to MPP processing

sites (Table 2), although its activities were lower than those of MPP (Supplemental Fig. 4).

Excess amounts of recombinant yeast c-MPP or d-MPP did not affect the substrate

specificity of RPP or enhance its activity (data not shown). It is likely that interaction

between RPP and MPP subunits are not necessary to cleave the peptides.

Regulation of preprotein processing by RPP - Next, we examined whether RPP

could cleave mitochondrial preproteins using mouse precursor of MDH (preMDH) and

bovine precursor of adrenodoxin (preADX), which contain different presequence lengths of

16 and 58 residues, respectively. RPP alone could not cleave either preMDH or preADX

(Fig. 4A). To our surprise, however, a stoichiometric mixture of RPP and yeast d-MPP

(RPP/d-MPP) processed preMDH, whereas a similar stoichiometric mixture of RPP and

yeast c-MPP did not. On the other hand, preADX was not cleaved by either RPP/d-MPP

or RPP1c-MPP. A functional association between RPP and d-MPP was established, since

d-MPP alone did not show any preprotein processing activities. However, the stable

RPP-d-MPP interaction was not demonstrated by d-MPP pull-down assays using the

His6-tag of RPP and affinity beads (data not shown), probably due to their weak association

with each other. To address which active sites were involved in the preMDH processing,

mutants of the catalytic glutamate residue in RPP and d-MPP (RPP* and d-MPP*,

respectively) were added to the processing assay either separately or together. As shown

in Fig. 4B, the processing of preMDH depended on the catalytic glutamate residue in

d-MPP, but not that in RPP, since RPP*/d-MPP cleaved the preprotein, whereas

RPP/d-MPP* and RPP*/d-MPP* did not, indicating that the active site of "d-MPP is

involved in the processing activity. Thus, RPP cooperates with d-MPP to recognize the

preprotein and activates the catalytic site of d-MPP. A single d-MPP never cleaves

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preproteins, and RPP is also inert for the cleavage. The 3D structure of MPP indicates that

the substrate around the cleavage point is accommodated in d-MPP. Taken together these, it

is (highly) likely that d-MPP/RPP complex processes preMDH at the correct site though

the actual peptide bond cleaved within preMDH is not identified heretofore.

GRL is required for cleavage of long presequences - Since RPP behaved as a

regulatory subunit toward preMDH processing, similar to c-MPP, we next investigated

why RPP/d-MPP was unable to cleave preADX. As mentioned above, there is a marked

difference in the presequence lengths between the two preproteins and RPP lacks a GRL

region. The GRL in yeast c-MPP was previously shown to play an essential role in

cleaving a synthetic preMDH peptide (23). Here, we analyzed whether an enzyme

comprised of GRL-deletion variants of yeast c-MPP"*c-MPPFGRL) and d-MPP (MPPFGRL)

could process the preproteins. MPPP

FGRL was able to cleave preMDH, albeit with a lower

processing activity than wild-type MPP, but showed inefficient preADX processing (Fig.

4C). An excess amount of c-MPPFGRL to d-MPP was null for the processing efficiency

(data not shown). A pull-down assay of c-MPPFGRL by d-MPP-His previously revealed a

stoichiometric association of c-MPP

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FGRL with d-MPP (23). These results therefore

suggest that the GRL region is required for the cleavage of long presequences, but does not

influence the association between the subunits. RPP/d-MPP complex behaved as

MPPFGRL at least for processing of preMDH and preADX. The reason for inability of

RPP/d-MPP complex to process preADX could be lack of GRL in RPP. To confirm

whether the GRL is sufficient for to cleave long presequences, we tried to constructs some

variants of the GRL-inserting RPP and to examine if this variant can process preADX.

However, the valiant proteins were misfolded due to the protein inclusion in E. coli and

they seem to be unstable during the refolding in vitro.

DISCUSSION

In the present study, we addressed the peptidase activity of a eubacterial homologue

of MPP and found a functional and evolutionary relationship between RPP and MPP. The

RPP cleaved basic peptides, including mitochondrial targeting presequence peptides, with

partial specificity for MPP cleavage sites. The most notable finding was that RPP was

able to activate eukaryotic d-MPP and subsequently render it able to process preproteins.

Thus, RPP retains not only the structural characteristics of the processing peptidases but

also the bifunctional hallmarks of the catalytic and regulatory subunits of MPP.

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Considering mitochondrial evolution on the basis of the endosymbiont theory, RPP appears

to have the closest remaining structural and functional hallmarks of a processing peptidase

in primitive mitochondria.

Although RPP is expressed in rickettsial cells, its functions in vivo remain unknown.

For instance, what is its function in rickettsiae and what is its physiological substrate?

One of the reasons for this lack of knowledge is the difficulty associated with elucidating

the exact actions of RPP in vivo, since rickettsiae can only live within other cells.

However, its actions in these cells may be speculated from the functions of the ymxG gene,

which encodes a type of M16 protease in the eubacterium Bacillus subtilis. The YmxG

protein is highly homologous to MPPs (26), and notably shares high degrees of similarity

(~60% identity and similarity) with RPPs and MPP-like proteins from the parasitic

eubacteria Mycobacterium leprae and Mycobacterium tuberculosis (27). Analyses of a

FymxG mutant strain revealed specific stimulation of the production of subtilisin (AprG),

the major serine protease secreted by B. subtilis (27). This phenomenon appears to arise

through negative regulation of aprE gene expression by YmxG, rather than through the

lack of YmxG proteolytic activity. As a consequence, Bolhuis et al. proposed two

possible models for the actions of YmxG (27). First, the protein could act as a repressor

via binding to the upstream sequence of the aprE gene or second, it could indirectly

modulate the activity of a transcriptional regulator of aprE, possibly through proteolysis.

Considering the homology of RPP to YmxG and the RPP peptidase/regulatory activities

revealed in the present study, RPP may play a key role in regulating protein expressions

through its protease activity. Even though the RPP functions in vivo remain unknown, the

fact that the eubacterial MPP-like protein YmxG is not essential for viability or cell growth

is interesting (27) when we consider the origin of these preprotein processing enzymes

according to mitochondrial endosymbiont evolution, as discussed below.

Genes for proteins containing zinc-binding HxxEH motifs characteristic of O38

proteases and sharing high degrees of similarity with MPPs were identified by sequence

searches of many bacterial genomes. Basic local alignment search tool (BLAST) (28)

searches of prokaryotic genomes revealed homologues to yeast MPP subunits

(Supplemental Table 1). Notably, there were nearly 500 putative proteins resembling

d-MPP in the 511 genomes of eubacteria examined, whereas proteins showing slight

homology to MPP with genetic significance were encoded in 29 genomes of archaebacteria.

In particular, BLAST searches of rickettsial genomes from a total of 20 species identified

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33 genes encoding d-MPP-like proteins, indicating that at least one RPP gene could be

carried on each rickettsial genome. Notably, no putative MPP-like proteins carrying

GRLs were found in the bacterial genomes. Considering the genomic distributions of

MPP-like genes described above, and since it is widely accepted that mitochondria

originated from the c-proteobacterial order Rickettsiales based on phylogenetic analyses

comparing the sequences of bacterial and mitochondrial genes (15-17, 29), MPP is unlikely

to have originated from proteins in the host cells of an archaebacterial ancestor, and is most

likely to have arisen from an RPP-like progenitor in a parasitic bacterium. During the

endosymbiotic evolution of mitochondria, the gene encoding the progenitor of MPP, which

may be nonessential, similar to ymxG in B. subtilis, could be transferred from the

endosymbiont to the host cell. At this stage, the nonessential gene conversion presumably

succeeded during mitochondrial evolution due to the maintained viability of the imperfect

symbiotic organelle. Alternatively, the genes may exist in both the endosymbiont and

host genomes under the predominant circumstance of the immature eukaryotic cells. In

any case, during or after the gene transfer, gain of the signal sequences and procurement of

the protein transport-processing system must be one of the critical stages toward the

success of endosymbiotic evolution in the primitive eukaryote cells.

Precise recognition of signal sequences by the protein translocation and processing

system must be one of the critical stages for the biogenesis of mitochondria, plastids and

hydrogenosomes, which are thought to have arisen from endosymbiotic bacteria.

Although it is unclear how the system was acquired during endosymbiotic evolution, the

central components of the mitochondrial translocases may be genetically converted from

eubacterial pore/channel-forming proteins and chaperones (30). The relationship between

MPP and RPP suggests the same situation, as mentioned above. Since MPP primarily

bears the traits of a parasitic bacterial peptidase, it appears that MPP and RPP have a

common progenitor in ancient parasitic bacteria. During the evolution stage from

endosymbiont to mitochondria, the progenitor gene was duplicated and the proteins were

converted into two distinct components of the processing enzyme, which are now c and

d-MPP (Supplemental Fig. 6). It appears that this protein dimerization was required for

the peptidase regulation and that gain of the GRL region was particularly involved in the

efficient processing of longer transport signal sequences, since these lengths have tended to

become elongated. Interestingly, the stromal processing peptidase that cleaves the signal

peptide of the plastidial protein precursor has an M16 protease active site and shares

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homology to MPPs, although it is active as a single polypeptide and carries no GRL region.

To decipher the complex histories of preprotein transport and processing systems in

endosymbiotic organelles, broad genetic investigations and biochemical analyses are

required for parasitic organisms and some lower eukaryotes, since it was recently reported

that Giardia mitosomes and Trichomonad hydrogenosomes share a common mode of

protein targeting (31).

ACKNOWLEDGMENTS

We thank Dr. Andersson and Dr. Kurland (University of Uppsala) for the genomic clones

including RP219 and Dr. Akiyama (Kyoto University) for E. coli strain AD293 transformed

with pKY206. This work was supported in part by Grants-in-Aid for Scientific Research

(to S. K.; No. 14658233) from the Ministry of Education, Science, Sports and Culture of

the Japanese Government.

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FIGURE LEGENDS

Fig. 1. Phylogenetic analysis of MPPs from various organisms, namely Homo sapiens,

Mus musculus, Solanum tuberosum, Saccharomyces cerevisiae and Trypanosoma brucei,

and RPPs from three Rickettsia species. The bootstrap values (>50%) are shown beside

each branch. The scale bar indicates the estimated sequence divergence per branch

length.

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Fig. 2. Structure comparison of RPPs and MPPs. A, Schematic diagrams of the primary

structures of RPP and the yeast S. cerevisiae MPP subunits. Light and dark gray regions

indicate c and d-MPPs, respectively, and their homologous regions in RPP. D, Crystal

structure of yeast MPP complexed with a synthetic presequence peptide. The structure is

shown as a ribbon diagram for the MPP subunits and a wire frame in a semi-translucent

space-filling model for the yeast COX IV(2-25) peptide. Quadrangles outlined with filled

and dotted lines include the active site and GRL regions, respectively.

Fig. 3. Alignments of the GRL region (upper) and active site region (lower) of RPPs and

MPP subunits. Identical and similar amino acids are indicated by (*) and (:) under the

alignments, respectively.

Fig. 4. Preprotein processing by RPP and MPP. A, Processing of preproteins by a

combination of RPP and d-MPP. Preprotein (preMDH or preADX) processing was

analyzed as described in the Materials and Methods. Lane 1, no enzyme; lane 2, yeast

MPP (0.1 og); lane 3, RPP (0.5 og); lane 4, RPP (1 og); lane 5, RPP/c-MPP (1 og each);

lane 6, RPP/d-MPP (1 og each). p, preprotein; m, mature protein. An asterisk indicates a

faint band under the band of preADX which may be mistranslated or degraded by proteases

during synthesis of the preprotein in in vitro translation system. B, Activation of d-MPP

by RPP. Each lane contains 0.2 og of each protein. Lane 1, RPP*; lane 2, RPP/d-MPP;

lane 3, RPP/d-MPP*; lane 4, RPP*/d-MPP; lane 5, RPP*/d-MPP*; lane 6, d-MPP. RPP*,

inactive mutant of RPP; MPP*, inactive mutant of d-MPP; p, preprotein; m, mature protein.

C, Processing of preproteins by a complex of c-MPPFGRL/d-MPP. Preprotein processing

was analyzed as described in the Materials and Methods. WT, wild-type MPP complex

(closed circles); FGRL,"c-MPPFGRL/d-MPP (open circles).

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Peptides* Peptide sequences and cleavage sites by RPP† Degradation‡

(%)Net charges§

A YGGFネLRRネIR PネKネLネK WDNQ 76.4 +4

B H SDAVFTDNネYTネRネLネRK QネMネAKK YLNSILN 36.2 +3

C INLK AネLAAネLAKネK IL 7.2 +3

D Ac-SYSMEHネFネR WGK PV-NH2 15.7 +1

E VNTPEHVVPYGLGSPR S <1.0 0

F CETQR TLLNGDLQTSI <1.0 -1

G LENLEAETAPLP <1.0 -3

H Pyr-GPWLEEEEEAYGWMDF-NH2 <1.0 -6

§Net charges are caluculated form acidic and basic amino acid residues in each peptide in the reaction condition.

Table 1. RPP cleaves basic peptides

*Peptide A ; dymorphin A, B ; vasoactive intestinal peptide, C ; mastoparan, D ; cMSH amide, E; bigendotheline, F ; peptide fragment of somatostatin recepr SSTR2A, G ; peptide fragment of o opioid receptorMOR1A, H ; Gastrin I.†Arrows denote RPP cleavage sites for peptides, which were able to be identified. Basic residues are indicatedin italics.‡Cleavage efficiencies are determined as in "Materials and Methods"

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Peptides* Peptide sequences and cleavage sites by RPP and MPP† Degradation by RPP‡

(%)

1 LSALARPVGAAネLRRSネニFSTSAQNNAKVA 59

2 MLAAKNILNRSSLSSSネニFRIATRLネQSTKVQGSA 39

3 LSRVAKRAネニFSSTVANP 20

4 MLRAAネLTTVRRGPRLSRネLネニLSAAATSAVPA 17

5 MFSネKLAHL ネQネRPAネVLSRGニVHSSVASA 17

6 MLSAARLQFAQGSVRRネニLTVSARDAPTKISTLA 9.5

7 GRWRLLVRPRAGAGGネLRGSRGPGLGGGAVATRTニLSV 7.3

8 LSLRQSIRFFKPATRT⊘LCSSTYLL <1

‡Cleavage efficiencies are determined as in "Materials and Methods"

Table 2. RPP cleaves mitochondrial presequence pepides

†Arrows (⊠) denote RPP-cleavage sites for peptides can be detected. MPP-cleavage sites areindicated by arrows (⊘)

*Peptide 1 ; mouse MDH2-28, 2; yeast HS771-32, 3; yeast MDH2-17, 4; mouse ALDH1-29, 5; humanOAT1-25, 6; yeast UCR21-32, 7; bovine ADX18-57, 8; yeast COXIV2-25.

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